Malaria remains one of the most prevalent and lethal human infectious diseases worldwide. Of the four species of malaria parasites that infect humans (Plasmodium falciparum, P. vivax, P. ovale, and P. malariae), P. falciparum and P. vivax are the most common. P. falciparum causes the most severe disease and almost all mortalities, whereas P. vivax causes repeated debilitating relapses. Failure to diagnose mixed infections and subsequent drug treatment for only the predominant species P. falciparum can allow the cryptic species P. vivax and others to rebound with severe clinical consequences. Conversely, if chloroquine is used for the treatment of P. vivax malaria in an area with high-level resistance to P. falciparum, the reduction of P. vivax infection may therefore increase the prevalence of P. falciparum infection and severe disease. Therefore, increasing the accuracy of diagnosis of mixed infections before treatment will be critical for the control of malaria infections. Furthermore, vaccination and therapeutic strategies to eradicate malaria worldwide should address mixed infections simultaneously. Failure to do so may lead to a tipping point that can change the dynamics of malaria transmission, mixed- species infections and severe clinical consequences at the population levels worldwide. Most studies on the dynamics of mixed-species infection were based on epidemiological observations using statistical-mathematical modeling. In order to move beyond phenomenological observation based on mathematical modeling to an immunological understanding of species and host-parasite interactions, we propose to take advantage of recent developments in both malaria genomic sequencing, proteomics, bioinformatics and high throughput proteome microarray generation/screening technologies to construct a blood stage proteome antigen array including all potential antigenic proteins that are expressed by blood stage parasites of P. falciparum and P. vivax. We will use this antigen array to study the antibody response profiles in humans with mixed-species infections to identify species-specific or cross-reactive antigens that are useful for development of effective and accurate diagnostics. This approach will provide new insights into the correlation between antibody profiles and disease states that will lead to the characterization of serological correlates of active and past infection, as well as antigen-specific protection. This information will be very valuable for the establishment of diagnostic algorithms and the development of rapid diagnostic testing kits for mixed species infections and evaluation tools for large vaccine trials in the endemic areas. This will also enable us to prioritize the most promising antigens for vaccine development to control mixed-species infections simultaneously. PUBLIC HEALTH RELEVANCE: Malaria remains one of the most prevalent and lethal human infectious diseases worldwide. Of the four species of malaria parasites that infect humans, P. falciparum and P. vivax are the most common. While P. falciparum causes the most severe disease and almost all mortalities, P. vivax causes repeated debilitating relapses. Failure to diagnose mixed infections and inadequate drug treatment can allow the cryptic species P. vivax and others to rebound with severe clinical consequences or may increase the prevalence of P. falciparum infection and severe disease in other areas. Here we propose to take advantage of recent advancements in high throughput proteomics to identify and test species-specific or cross-reactive antigens that are essential for development of robust and accurate diagnostics and for designing safe and effective vaccines against malaria. The proposed study will have a tremendous sanitary and economic impact throughout the world.